Polysialic Acid Derivatives

ABSTRACT

A polysialic acid compound is reacted with a hetero-bifunctional reagent to introduce a pendant functional group for site-specific conjugation to sulfhydryl groups, for instance side chains of cysteine units in drugs, drug delivery systems, proteins or peptides. The functional group is, for instance, an N-maleimide group.

This application is a continuation pursuant to 35 U.S.C. §120 and claimsthe benefit of priority to U.S. application Ser. No. 13/650,048, filedon Oct. 11, 2012, a continuation application that claims priority toU.S. application Ser. No. 12/717,073, filed Mar. 3, 2010, now abandoned,a divisional application that claims priority to U.S. application Ser.No. 10/568,111, filed Jul. 13, 2006, now U.S. Pat. No. 7,691,826, whichis the U.S. national phase application pursuant to 35 U.S.C. §371 ofPCT/GB2004/003488, filed Aug. 12, 2004, which claims the benefit ofpriority to European Application No. 03254988.3, filed Aug. 12, 2003,and European Application No. 03255200.2, filed Aug. 21, 2003; each ofwhich is hereby incorporated by reference in its entirety.

The present invention relates to polysialic acid derivatives which areuseful for conjugation to drugs, proteins and peptides, or to drugdelivery systems such as liposomes, having sulfhydryl groups, as well asto conjugated products process for synthesising the derivatives and theconjugates and novel synthetic intermediates.

The extended presence of drugs either within the vascular system or inextravascular use is often a pre-requisite for their optimal use. Manyantibiotics and cytostatics for instance, as well as a variety oftherapeutic peptides and proteins, and liposomes are removed from thecirculation prematurely and before effective concentrations in targettissues can be achieved. The half lives of a number of short-livedproteins (for instance enzymes, cytokines, etc) have been augmented byconjugating these to poly(ethylene glycol). It appears that PEGmolecules prolong the circulation time of proteins and particles byforming a cloud around their surface, thus sterically hinderinginteraction with factors responsible for their clearance. However PEG isnon-biodegradable and accumulation of PEGylated proteins intracellularymay be undesirable especially on chronic use [Bendele, A., Seely, J.,Richey, C., Sennello, G., Shopp, G., Renal tubular vacuolation inanimals treated with polyethylene-glycol conjugated proteins,Toxicological sciences, 42 (1998) 152-157; Conyers, C. D., Lejeune, L.,Shum, K., Gilbert, C., Shorr, R. G. L, Physiological effect ofpolyethylene glycol conjugation on stroma-free bovine hemoglobin in theconscious dog after partial exchange transfusion, Artificial organ, 21(1997) 369-378].

We have described the conjugation of a polysaccharide comprising 2→8 andor 2→9 (e.g. alternating 2→8 and 2→9) linked sialic acid unitsconjugated to proteins to increase their half life, reduce theirimmunogenicity/antigenicity or increase the stability of a variety ofproteins. In WO92/22331, polysialic acids are reacted with a model drugand shown to extend the half life in the circulation of mice. In Cell.Mol. Life Sci. 57 (2000) 1964 to 1969 and in Biotechnol. Genet. Eng.Rev. 16 (1999) 203 to 215, Gregoriadis et al. describe the conjugationof polysialic acids to asparaginase and catalase, and show that theclearance rates from circulation reduced whilst enzyme activity wasretained. We have also polysialylated insulin (Biochim. Biophys. Acta1622 (2003) 42-49 and shown it to be active. We have also polysialylatedinterferon (AAPS Annual meeting 2002, Toronto, Canada, M1056). We havealso polysialylated antibody fragment Fab (Epenetos, A. et al.,Proceedings of ASCO (Clinical Pharmacy) 21 (2002) 2186).

In all of these publications, polysialic acid is rendered reactive, bygenerating an aldehyde group at the non-reducing end by oxidation of the7, 8-vicinal diol moiety with sodium periodate. The aldehyde group wasthen reacted with primary amine groups on proteins, generally assumed tobe epsilon-amino groups of lysine moieties of the protein or N-terminalamine groups. The reaction forms a Schiff base which is reduced bycyanoborohydride to a secondary amine.

In WO-A-01/87922 we also suggest that derivatisation with othermolecules could be carried out in the presence of denaturant to achieveincreased levels of derivatisation. Examples of other derivatisingagents are polyethylene glycol compounds. Activated PEG compounds suchas tresyl-PEG and succinimidyl succinate ester of PEG were mentioned.The examples used succinimidyl succinate activated PEG, which isbelieved to react with amine groups.

PEG derivatives having functional groups for coupling to thiol groupsare commercially available. The functional groups may be maleimide,vinyl sulfone, iodoacetamide or orthopyridyl disulphide groups. Sincethese reagents react specifically with cysteines, and since proteinshave fewer cysteines on their surfaces than lysine groups, thederivatisation is more controllable. Furthermore, in the absence of afree cysteine in a native protein, one or more free cysteines may beadded by genetic engineering. The advantage of this approach is that itmakes possible site-specific derivatisation at areas on the proteinwhich will minimise a loss in biological activity.

PEGylated proteins have been found to generate anti PEG antibodies thatcould also influence the residence time of the conjugate in the bloodcirculation (Cheng, T., Wu, M., Wu, P., Chern, J, Roffer, S R.,Accelerated clearance of polyethylene glycol modified proteins byanti-polyethylene glycol IgM. Bioconjugate chemistry, 10 (1999)520-528). Despite, the established history of PEG as a parenterallyadministered polymer conjugated to therapeutics, a better understandingof its immunotoxicology, pharmacology and metabolism will be required(Hunter, A. C; Moghimi, S. M., Therapeutic synthetic polymers: a game ofRussian Roulette. Drug Discovery Today, 7 (2002) 998-1001; Brocchini,S., Polymers in medicine: a game of chess. Drug Discovery Today, 8,(2003) 111-112).

It would be useful for modification by polysialic acid to be targetedtowards thiol (sulfhydryl) groups. It would also be desirable for theefficiency of derivatisation by sialic acid to be increased, theprocesses described in our prior art mentioned above requiring highexcesses of active polysialic acid. It would also be desirable to avoidthe use of cyanoborohydride.

According to the present invention there is provided a novel compoundcomprising a polysaccharide having a moiety linked at one or eachterminal unit which includes a functional group selected fromN-maleimido groups, vinysulphone groups, N-iodoacetamide groups andorthopyridyl disulphide groups.

The terminal unit to which the moiety is linked may be at thenon-reducing end of the polysialic acid or at the reducing end of thepolysialic acid. Generally the terminal sialic acid unit has beensubjected to a preliminary chemical reaction to generate usefulfunctional groups to which a maleimide-group containing reagent may belinked. We have found it convenient to use the chemistry disclosed inour earlier publications in which an aldehyde group is generated, as apreliminary step to generate the functional group via which themaleimide moiety may be linked.

In our earlier publications mentioned above, it is the non-reducingterminal unit which is converted into an aldehyde moiety by oxidation ofthe 7, 8-vicinal diol moiety with sodium periodate to form the carbon7-aldehyde compound. This is an appropriate reaction for the presentinvention.

As an alternative, it is possible to provide the aldehyde moiety at thereducing terminal unit. In this case, it is preferred (but is notessential) to carry out a preliminary step in which the non-reducing endis deactivated, by preliminary oxidation and reduction steps. A firstreduction step converts the ketal unit at the reducing end into areduced ring opened form, having vicinal diols. The vicinal diols aresubsequently oxidised using sodium periodate to form an aldehyde moietyat the carbon atom previously forming the 7-carbon of the reducingterminal unit. Where the non-reducing terminal glycosyl (usually sialicacid) is not deactivated by a preliminary oxidation step, the terminalunit will be simultaneously activated.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an SOS-PAGE gel of triplicate samples of the conjugate ofcolominic acid maleimide (CAM) with Fab and relevant controls.

FIG. 2 shows an SOS-PAGE gel of the conjugate of colominic acidiodoacetate (CAI) with P-galactosidase and of CAM with p-galactosidase.

FIG. 3 shows SOS-PAGE gels of conjugates of P-galactosidase withalternate colominic acid derivatives.

DETAILED DESCRIPTION

In the invention, the moiety which includes the functional group, may belinked directly to the polysialic acid unit or, more conveniently, maybe linked via a difunctional organic group, such as an alkane diylgroup, an arylene group or an oligo(alkyleneoxy) alkane group, oralternatively an oligo peptidyl group. The linkage to the polysialicacid (from the linker or the moiety including the functional group maybe a secondary amine, a hydrazone, an alkyl hydrazine an ester or apeptidyl group. The moiety may be generated by reaction of a polysialicacid substrate with a heterobifunctional reagent. The process form afurther aspect of the invention.

Reagents useful for introducing the selected functional groups arecommercially available. A compound which will introduce a maleimidegroup onto an amine moiety without introducing any additional linkermoiety is N-methoxy-carbonyl-maleimide. Generally the reagents include asecond functional group for reaction with a group on the polysialic acidwhich may be the aldehyde group described above, or an amine group.Suitable second functional groups include N-hydroxy succinimide estersand their sulfosuccimide analogues and hydrazides. Preferably thecompound is a N-maleimido-alkanoic acid hydrazide or anN-maleimidoarylalkanoic acid hydrazide i.e. a compound having thegeneral formula

X—R—Y

-   -   in which:    -   X is a N-maleimido, N-iodoacetamido, S-vinylsulphonyl or        S-orthopyridyldisulphide group,    -   R is alkane-diyl, arylene or aralkylene alkarylene,        alkylene-oxaalkylene, or alkylene-oligooxa-alkylene or        alkyl-oligopeptidyl-alkyl group; and    -   Y is a hydrazide, amine or N-hydroxysuccinimide group.        Preferably R is C₁₋₆ alkanediyl, C₂₋₃-alkyl-oxa-C₂₋₃-alkylene,    -   C₂₋₃ alkyloligo(oxa-C₂₋₃ alkylene), or C₂₋₆ alkylene phenyl.

Preferably X is N-maleimido or orthopyridyldisulphide. Preferably Y is ahydrazide or a hydroxyl succinimide. Compounds which may be reacted withan aldehyde group, and which include a linker moiety and introduce amaleimide group include N-[β-maleimidopropionic acid]hydrazide and4-(4-N-maleimidophenyl)butyric acid hydrazide. The hydrazide groupreacts with the aldehyde to form a stable hydrazone group. A suitableheterobifunctional compound which includes an oligo(ethyleneoxy)ethylene group is a compound comprising a polyethylene glycol(poly(ethyelenoxy)) group with, at one end, N-hydroxy succinimide (NHS)group and at the other end the functional group. The NHS group reactswith amine groups to form stable amide linkages. Heterobifunctionalpolyethyleneglycols with NHS at one end and either vinylsulphone ormaleimide at the other end are available. Other examples ofheterobifunctional reagents include, 3-(2-pyridyldithio)propionylhydrazide, N-succinimidyl-3-[2-pyridyldithio]propionate,succinimidyl-H-[N-maleimidomethyl]cyclohexane-1-carboxylate),m-maleimidobenzoyl-N-hydroxysuccinimide ester,N-succinimidyl-[4-iodoacetyl]amino benzoate,N-[gamma-maleimidobutyryloxy]succinimide ester,N-[epsilon-maleimidocaproyloxy]succinimide ester and N-succinimidyliodoacetate. Other reagents are available from Pierce Biotechnology andShearwater Corporation (polyethylene glycol-based).

Sialic acids (also known as nonulosonic acids) are members of a familyof amino containing sugars containing 9 or more carbon atoms. The mostimportant of the sialic acids is N-acetylneuraminic acid (also known as5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-nonulosonic, lactaminicacid and O-sialic acid) which has the formula:

Polysialic acids may be linked 2→8 and/or 2→9, usually in theα-configuration. In some polysialic acids the linkages are alternating2→8 and 2→9. The invention is also of utility for heteropolymericpolysaccharides comprising glycosyl units other than sialic acid units.

Polysialic acids are generally found to be non-toxic and substantiallynon-immunogenic. Furthermore the biodegration units, sialic acid, is notknown to be toxic and, indeed sialic acids are widely found in animalproteins and cells, including blood cells and circulating proteins.

Polysaccharide compounds containing many sialic acid units arepolysaccharides produced by Escherichia coli, Moraxella nonliquifaciens,Pasteurella aeroginosis and Neisseria meningitidis or derivativesthereof. For instance colominic acid derived (by hydrolysis to shortenthe chain lengths) from E. coli K1 comprises α 2→8 linked sialic acidunits. Polysaccharide from E. coli K92 strain comprises alternating 2→8and 2→9 linked sialic acid units. Polysaccharide C of N. meningitidisgroup C has 2→9 linked sialic acid units.

One group of polysaccharide compounds which has been found to be ofparticular utility in the invention is group B polysaccharides. Thesecompounds are produced by N. meningitidis, M. nonliquifaciens, P.aeroginosis A2 and E. coli K1. These compounds comprise a polysaccharidecomponent comprising sialic acid residues and a phospholipid component.The sialic acid residues are linked a 2→8 the naturally occurringpolymer consisting of about 200 residues. Some of the glycolipidmolecules, especially the high molecular weight compounds appear to havea covalently attached phospholipid at the reducing end of thepolysaccharide component.

It is preferable for the bacteria from which the polysaccharide compoundis derived to be non-pathogenic for convenience during production. It isparticularly suitable therefore for the polysaccharide to be derivedfrom a non-pathogenic strain of E. coli such as E. coli K92 or,preferably, K1 which is non-immunogenic. E. coli K92 and K1 isolates arewell-known and any such type of any such strains can be used as sourcesof suitable polysaccharide. Preferably the polysialic acid should haveat least 2, preferably at least 5, more preferably at least 10, forinstance more than 50 sialic acid units.

According to the invention, there is also provided a conjugate of aprotein and the novel activated polysaccharide. The novel compoundcomprises a protein with at least one cysteine unit and, linked througha thioether bond to the side chain of a cysteine unit, a polysialic acidthrough a moiety joined at one or both terminal unit of the polysialicacid.

Where the polysialic acid derivative was a N-maleimido group the moietywill include a N-succinimidyl group, with the thioester linked at theα-carbon atom. Preferably the moiety also comprises a secondary amine, ahydrazone or an amide bond.

There is also provided in the invention a new process in which apolysialic acid is reacted with a heterobifunctional reagent having afirst functional group for reaction with sulfhydryl groups and a secondfunctional group different to the first group whereby the said secondfunctional group reacts with a terminal unit of the polysialic acid toform a covalent bond therewith and form a capable of reaction with asulfhydryl group functional polysialic acid.

In one embodiment the second functional group is a nucleophilic group,preferably hydrazine. This is of particular utility where the polysialicacid comprises an aldehyde group in the terminal unit whose carbonylgroup is attacked by the nucleophilic group.

In another embodiment of the process the second functional group iselectrophilic such as an N-alkoxyl carbonyl-imide such asN-hydroxysuccinimide ester or sulphosuccinimide ester, or carbodiimide.The terminal group in such cases is preferably nucleophilic such asamine.

In the process it is preferred that the reagent comprises a bifunctionalorganic group linking the first and second functional groups. Preferablythe bifunctional organic group is selected from a C₂₋₁₈-alkanediylgroup, an arylene group, an oligo peptide and an oligo(alkoxy)alkylgroup.

Examples of suitable reagents are given above.

Most usefully the process involves a subsequent step in which thefunctional polysialic acid is reacted with a polypeptide or a proteinhaving at least one free and unprotected Cys unit whereby the functionalgroup forms a thioether linkage with the thiol group of a Cys unit toform a polysialyated polypeptide or protein. The process is particularlysuitable where the protein contains a single Cys unit, wherebysite-controlled derivatisation is achieved.

The invention is illustrated in the accompanying examples.

EXAMPLE 1 1.1 Synthesis

Three separate preparations were carried out as follows:

Colominic acid aldehyde (CAO) produced according to WO-A-9222331 (100mg, 4.4×10⁻⁶ mol) was dissolved into 500 μl 0.1 M sodium acetate, tothis 5 molar equivalents of N-[β-Maleimidopropionic acid]hydrazide (6.5mg, 2.2×10⁻⁵ mol) was added. This mixture was then vortex mixed andwrapped in foil and allowed to incubate at 37° C. for 2 h on a rotarymixer. The polymer was then precipitated by the addition of 2 volumes(1.0 ml) of ethanol. The precipitate was collected by centrifugation(13,000 rpm 2 min) in a bench top microcentrifuge. The supernatant wasdiscarded and the pellet dissolved in 500 μl 0.1 M acetate. This processwas repeated a further 2 times and the final pellet dissolved indeionised water and freeze dried overnight.

1.2 Assay for Maleimide Content

In this assay cysteine is reacted with the maleimide on the polymerpreventing further reaction with Ellman's Reagent(5,5′-dithiobis(2-nitrobenzoic acid)) which contains a disulphide whichforms an intense yellow colour when it is substituted for a thiol notadjacent to an aromatic ring. Thus maleimide content can be calculatedby measuring the inhibition of reaction between cysteine and Ellman'sreagent assay.

First an initial stock of cysteine at 12×10⁻³ M (0.145 mg/ml) wasprepared in PBS. In a clean microtitre plate 100 μl volume doublingdilutions from 12×10⁻³ M to 0.375×10⁻³ M were made from row B to row H.In row A 100 μl PBS was used as a zero standard. Samples of CA andCA-maleimide (CAM) were prepared at 5 or 10 mg/ml and 100 μl of eachsample added to duplicate columns of the cysteine dilutions. In one set100 μl PBS without any CA was added as a control. The plate was coveredand allowed to incubate at 37° C. for 1 h. After this time 20 μl ofEllman's reagent (4 mg/ml) was added to each well and the plateincubated in the dark at room temperature for 15 min. Absorbance was themeasured in wells at 405 nm. Standard curves were then plotted for thesamples and the amount of maleimide present calculated from inhibitionof the signal.

1.3 (Intentionally Omitted) 1.4 Thiolation of FAb and Conjugation to CAM

In the first step a thiol group is introduced into a model protein bythiolation of an amine of lysine.

Ovine FAb (anti Desipramine /Norityrptaline, 4 mg, 7.2×10⁻⁸ mol) wasdissolved in 0.25 ml PBS+10 mM EDTA to this was added 0.498 mg2-iminothiolane (2-IT or Traut's reagent 50 mol equiv 3.6×10⁻⁶ mol) in0.25 ml of the same buffer. The tube was wrapped in foil and left toincubate stirring end over end for 1 h at 37° C.

Thiolated Fab was purified from free 2-IT (Traut's reagent) by gelfiltration (PD-10) and 0.5 ml fractions assayed for presence of protein(BCA assay) or thiol (Ellman's assay). The first eluting peak containingboth was pooled and protein and thiols quantified.

1.5 Conjugation of Fab-Thiol to CAM

To thiolated FAb (3.6 mg, 6.6×10⁻⁸ mol) in 2 ml PBS/EDTA 22.5 mg CAM wasadded (9×10⁻⁷ mol, 15 molar equiv). The tube was sealed and allowed toincubate at 37° C. for 1 h whilst gently mixing. The resulting conjugatewas then purified according to accepted protocols to remove free CAM.Both CA and protein content were assayed on the conjugate to calculateconjugation ratio.

Control reactions were carried out with CA as a negative control.

Several batches of CAM-Fab were prepared with various degrees ofthiolation but maintaining the 15:1 ratio of CAM: Fab. Results are shownin table 1 below:

TABLE 1 Thiol per FAb Conjugate ratio (CA:Fab)  1  0.53:1  2  0.9:1  5(triplicate reaction) 1.925:1 +/− 0.19:1 Fab 10  3.51:1

FIG. 1 shows an SDS-PAGE gel of triplicate samples and relevant controls

1.6 Conclusion

The results show that in all control wells (samples of thiolated FAb)the migration of the sample is similar to that for fresh FAb (below thatof the 50 kDa marker) indicating no cross linking of FAb moleculesduring the process of conjugation. In the replicate lanes there isconsiderable band broadening with maximum intensity between the 98 and250 kDa markers which typically indicates an increase in mass which isindicative of a of polysialylated product.

1.7 Conjugation of CAM to Beta Galactosidase

To E. coli β-galactosidase (β-gal: 5.0 mg, 4.3×10⁻⁸ mol) in 1 ml PBS 15mg CAM was added (6.59×10⁻⁷ mol, 15 molar equiv). The tube was sealedwrapped in foil and allowed to incubate at room temperature for 1 hwhilst gently mixing. The resulting conjugate was analysed by SDS pageand then purified according to accepted protocols to remove free CAM.Samples were assayed for polymer and protein content as outlinedelsewhere.

Control reactions were carried out with CA as a negative control.

1.8 Assay for Enzyme Activity

Standards from 60 μg/ml to 3.75 pg/ml of fresh β-galactosidase wereprepared in PBS. Sample of CAM-β-gal were diluted to 60 μg/ml in thesame buffer. Enzyme activity of the conjugates was measured as follows:In a microtitre plate, to 100 μl of sample or standard was added 100 μlof All-in-One β-gal substrate (Pierce). The plate was incubated at 37°C. for 30 min and absorbance read at 405 nm. A calibration curve wasprepared from the standards and the activity of the samples calculatedfrom the equation for the linear regression of the curve.

1.9 Results

From the protein and polymer assays the conjugation ratio was determinedto be 1.23 CAM:1 β-Gal. There was also a corresponding increase inapparent molecular mass from the SDS page of the samples (FIG. 2).Enzyme activity in the purified sample was calculated to be 100.4%compared to the free enzyme.

EXAMPLE 2 Synthesis Route 2

Step 1 Amination of CA-Aldehyde (CHO)

Step 2 Introduction of Maleimide Ring

Synthesis

2.1 Step 1 Amination of Oxidised CA

Oxidised colominic acid at (CAO) 10-100 mg/ml was dissolved in 2 ml ofdeionised water with a 300-fold molar excess of NH₄Cl, in a 50 ml tubeand then NaCNBH₃ (5 M stock in 1 N NaOH(aq)) was added at a finalconcentration of 5 mg/ml. The mixture was incubated at room temperaturefor 5 days. A control reaction was also set up with colominic acid (A)instead of CAO. Product colominic acid amine derivative was precipitatedby the addition of 5 ml ice-cold ethanol. The precipitate was recoveredby centrifugation at 4000 rpm, 30 minutes, room temperature in abenchtop centrifuge. The pellet was retained and resuspended in 2 ml ofdeionised water, then precipitated again with 5 ml of ice-cold ethanolin a 10 ml ultracentrifuge tube. The precipitate was collected bycentrifugation at 30,000 rpm for 30 minutes at room temperature. Thepellet was again resuspended in 2 ml of deionised water andfreeze-dried.

2.2. Assay for Amine Content

The TNBS (picrylsulphonic acid or 2, 4, 6-tri-nitro-benzene sulphonicAcid) assay was used to determine the amount of amino groups present inthe product.

In the well of a microtitre plate TNBS (0.5 μl of 15 mM TNBS) was addedto 90 μl of 0.1 M borate buffer pH 9.5. To this was added 10 μl of a 50mg/ml solution of CA-amide the plate was allowed to stand for 20 minutesat room temperature, before reading the absorbance at 405 nm. Glycinewas used as a standard, at a concentration range of 0.1 to 1 mM. TNBStrinitrophenylates primary amine groups. The TNP adduct of the amine isdetected.

Testing the product purified with a double cold-ethanol precipitationusing the TNBS assay showed close to 90% conversion.

2.3 Maleimidation of CA-Amine

CA-Amine (17 mg) was dissolved in 1 ml deionised water, to this wasadded 6 mg methoxy-carbonyl-maleimide (MCM). The mixture was left toreact at room temperature for 30 min. To the sample 100 μl water and 200μl acetonitrile was added and then incubated at room temperature for 4h, after which 300 μl CHCl₃ was added, the tube shaken and the aqueousfraction collected. Then the fraction was purified over a PD-10 columnto remove small molecules. The eluate was freeze dried and assayed formaleimide content. The molar concentration of maleimido was 44 mol %.

EXAMPLE 3 Preparation of Iodoacetate Derivative of Colominic Acid (CAI)

3.1 Synthesis

To 40 mg colominic acid amine (85 mol % amine) as (described in Example2.1) dissolved in 1 ml of PBS pH 7.4 was added 5 mg of N-succinimidyliodoacetate (SIA). The mixture was left to react for 1 h at 37° C.,after which excess SIA was removed by gel filtration over a 5 mlHIGHTRAP™ Desalting column (AP Bioscience) eluted with PBS. 0.5 mlfractions were collected from the column and samples from each fractiontested for colominic acid content (resorcinol assay) and reactivity withcysteine indicating Iodide (Ellman's Assay). Fractions positive for bothiodide and CA were pooled.

3.2 Conjugation of CAI to β-Galactosidase

To E. coli β-galactosidase (5.0 mg, 4.3×10⁻⁸ mol) in 1 ml PBS 15 mg CAIwas added (6.59×10−7 mol, 15 molar equiv). The tube was sealed wrappedin foil and allowed to incubate at room temperature for 1 h whilstgently mixing. The resulting conjugate was analysed by SDS page and thenpurified according to accepted protocols to remove free CAI. Sampleswere assayed for polymer and protein content as outlined elsewhere.

Control reactions were carried out with CA as a negative control. Allsamples were analysed for β-gal activity as example 1.8 above.

3.3 Conclusions

Fractions 3-6 were positive for both polymer and iodoacetate and werepooled. The SDS page (4-12% Bis/Tris gel; FIG. 2) showed an increase inapparent molecular mass for samples incubated with the iodoacetamidederivative but not with control polymer. From the protein and polymerassays the conjugation ratio was determined to be 1.63 CAI:1 β-gal.β-gal activity was calculated to be 100.9% for the conjugated sample,compared to the free enzyme.

EXAMPLE 4 4.1 Synthesis: Non-Reducing End Activation with4(4-N-maleimidophenyl)butyric acid hydrazide (CA-MBPH) and3-(2-pyridyldithio)propionyl hydrazide (CA-PDPH)

The preparations were made as follows:

Colominic acid aldehyde (CAO; 22.7 kDa, Camida, Ireland) producedaccording to WO-A-9222331 (73 mg for MBPH, tube A; 99.3 mg for PDPH;tube B) was dissolved individually into 800 μl 0.1 M sodium acetate (pH5.5). To tube A 15 mg of MBPH (15:1 linker:CA ratio) dissolved in 200 μlof DMSO was added. To tube B 15 mg (15:1 linker:CA ratio) of PDPHdissolved in 200 μl of DMSO was added. The pH was adjusted to 5.5 ineach case for each reaction vessel. These mixtures were then vortexmixed and wrapped in foil and allowed to incubate at 37° C. for 2 h onan orbital mixer. Each polymer solution was purified by gel filtration(PD 10 column) eluting with PBS pH 7.4 and the 1 ml fraction containingCA (by resorcinol assay) collected. These sample were freeze driedovernight and assayed for maleimide content as described in example 1.2.

4.2 Synthesis: Reducing End Activation with4(4-N-maleimidophenyl)butyric acid hydrazide (MBPH-CA),(2-pyridyldithio)propionyl hydrazide (PDPH-CA) andN-β-Maleimidopropionic acid hydrazide)hydrazide (BMPH-CA)

The preparations were carried out as follows all at molar ratios of 25:1linker to CA:

Colominic acid (CA; 22.7 kDa; 73 mg for MBPH, tube A; 99.3 mg for PDPH;tube B and 76.6 mg for BMPH; tube C) was dissolved individually into 800μl 0.1 M sodium acetate (pH 5.5) separately. To tube A, 25 mg of MBPH(dissolved in 200 μl of DMSO), to tube B 25 mg of PDPH (dissolved in 200μl of DMSO) and to vial C 25 mg of BMPH (dissolved in 200 μl of sodiumacetate buffer) were added. The pH of the reaction mixture was adjustedto 5.5. Each mixture was then vortex mixed, wrapped in foil and allowedto incubate at 37° C. for 72 h on an orbital mixer. Each polymersolution was purified by gel filtration (PD 10 column) eluting with PBSpH 7.4 and the 1 ml fraction containing CA (by resorcinol assay)collected. These sample were freeze dried overnight and assayed formaleimide content as described in example 1.2.

4.3 Results

The molar concentration of maleimido was 49.0 and 35.0 mol % for MBPHand PDPH respectively on the non-reducing (highly reactive) end. Themolar concentration of maleimido was 41.5, 32.5 and 48.3 mol % for MBPH,PDPH and BMPH respectively on the reducing end (weakly reactive). Thevalues on the reducing end are average of two values in each case.

4.4 Conjugation of β-galactosidase (β-gal) to Maleimide ActivatedColominic Acids (Reducing End and Non-Reducing End)

To β-gal (1.0 mg; 1 ml in PBS) in separate tubes, a 15 molar excess ofeach maleimide activated CA (MBPH, PDPH and BMPH on non-reducing orreducing end, from above examples) was added separately. Each tube wassealed and allowed to incubate at 37° C. for 1 h whilst gently mixing.The resulting conjugate was then purified according to acceptedprotocols to remove free activated polymer. All samples were analysed bySDS PAGE and for β-gal activity as in example 1.8

4.5 Results

The results (FIG. 3) show that in all control well (with β-gal) themigration of the sample is similar to that for fresh β-gal. In theconjugate lanes there is considerable band broadening with maximumintensity between the 98 and 250 kDa markers which typically indicatesan increase in mass which is indicative of a of polysialylated-β-gal.β-gal activity was calculated to be 91.0-106% for the conjugatedsamples.

1. A polysaccharide having at least two sialic acid units linked 2.8and/or 2.9 to one another, comprising a compound having the generalformula:X—R—Y in which: X is a N-maleimido, N-iodoacetamido, S-vinylsulphonyl orS-orthopyridyldisulphide group, R is alkane-diyl, arylene or aralkylenealkarylene, alkylene-oxaalkylene, or alkylene-oligooxa-alkylene oralkyl-oligopeptidyl-alkyl group; and Y is a hydrazide, amine orN-hydroxysuccinimide group.
 2. The polysaccharide of claim 1, wherein Ris C₁₋₆ alkanediyl, C₂₋₃-alkyl-oxa-C₂₋₃-alkylene, C₂₋₃alkyl-oligo(oxa-C₂₋₃ alkylene), or C₂₋₆ alkylene phenyl.
 3. Thepolysaccharide of claim 1, wherein X is N-maleimido ororthopyridyldisulphide.
 4. The polysaccharide of claim 1, wherein Y is ahydrazide or a hydroxyl succinimide.
 5. The compound of claim 1, whereinthe polysaccharide comprises sialic acid units.
 6. The compound of claim1, wherein the polysaccharide is colominic acid.
 7. The compound ofclaim 1, wherein the polysaccharide comprises glycosyl units other thansialic acid units.
 8. The compound of claim 1, wherein thepolysaccharide comprises a polysaccharide component and a phospholipidcomponent.
 9. The compound of claim 8, wherein the phospholipidcomponent is covalently attached to the reducing end of thepolysaccharide.
 10. The compound of claim 1, wherein the polysaccharideis a B polysaccharide.
 11. The compound of claim 1, wherein thepolysaccharide comprises at least 2, at least 5, at least 10, or atleast 50 sialic acid units.
 12. A polysaccharide protein conjugatecomprising the polysaccharide of claim 1 linked through a thioether bondto the side chain of a cysteine unit of the protein.
 13. Thepolysaccharide protein conjugate of claim 12, wherein the polysaccharideis linked through a moiety joined at one or both terminal units of thepolysaccharide.
 14. The polysaccharide protein conjugate of claim 12,wherein the protein contains a single Cysteine.
 15. The polysaccharideprotein conjugate of claim 12, wherein the polysaccharide comprisessialic acid units.
 16. The polysaccharide protein conjugate of claim 12,wherein the polysaccharide is a B polysaccharide.
 17. The polysaccharideprotein conjugate of claim 12, wherein the polysaccharide is linkedthrough an N-succinimidyl group with the thioester linked at theα-carbon atom.
 18. The polysaccharide protein conjugate of claim 12,wherein the moiety comprises a secondary amine, a hydrazine, or an amidebond.
 19. A heterobifunctional reagent having a first functional groupfor reaction with sulfydryl groups and a second functional groupdifferent from the first group whereby the second functional groupreacts with a terminal unit of a polysialic acid to form a covalentbond.
 20. The heterobifunctional reagent according to claim 19, whereinthe second functional group is a hydrazine or an N-alkoxylcarbonyl-imide.